Extending the capabilities of intact-mass analyses to monoclonal immunoglobulins of the E-isotype (IgE).
Extending the capabilities of intact-mass analyses to monoclonal immunoglobulins of the E-isotype (IgE).
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DOI：
10.1080/19420862.2022.2103906
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发表时间：
2022-01
期刊：
影响因子：
5.3
通讯作者：
Kaltashov, Igor A.
中科院分区：
文献类型：
--
作者：
Yang, Wenhua;Ivanov, Daniil G.;Kaltashov, Igor A.
关键词：
质谱（MS）已成为单克隆抗体（mAb）的结构表征和质量控制中不可或缺的工具。完整质量分析是一个特别有吸引力的选择，它提供了一种强大且具有成本效益的方法，不仅可以确认蛋白质的结构完整性，还可以探测其与治疗靶点的相互作用。在一定程度上，这种成功可归因于IgG分子表现出的相对适度的糖基化水平，这限制了它们的结构异质性，并能够在完整分子水平上进行直接的质量测量。最近对扩大mAb库以包括其他类别的免疫球蛋白的兴趣激增，这使得将IgG定制的实验策略适应其他类别的抗体的努力受到重视，但其显著更高的糖基化水平可能会产生不可逾越的障碍。在这项工作中探索的单克隆鼠IgE抗体提供了一个具有挑战性的模型系统，因为它的糖基化水平超过了常规IgG mAb的9倍。商业样品，其中包括各种IgE片段，产生一个很差的解决离子信号在完整的质量测量，从中可以提取很少有用的信息。然而，耦合MS测量与有限的电荷减少选择聚阳离子物种在气相中产生明确定义的电荷梯，离子质量和电荷都可以很容易地确定。测量揭示了完整IgE分子内糖基化程度的显著变化，以及商业IgE样品中低分子量杂质的存在。此外，单克隆IgE与其抗原（卵清蛋白）的孵育引起具有不同化学计量的复合物的形成，其也可以使用天然MS、气相中的有限电荷减少和数据拟合程序的组合来唯一地鉴定。这项工作表明，经过适当的修改，完整的质量分析测量可以成功地应用于单克隆抗体超越IgG同种型，提供了丰富的信息，不仅对完整的IgE分子的质量分布，而且其大规模的构象完整性，其共价结构的完整性，以及它们与抗原的相互作用。
Mass spectrometry (MS) has become an indispensable tool in structural characterization and quality control of monoclonal antibodies (mAbs). Intact-mass analysis is a particularly attractive option that provides a powerful and cost-effective means to not only confirm the structural integrity of the protein, but also probe its interactions with therapeutic targets. To a certain extent, this success can be attributed to relatively modest glycosylation levels exhibited by IgG molecules, which limits their structural heterogeneity and enables straightforward mass measurements at the intact molecule level. The recent surge of interest in expanding the repertoire of mAbs to include other classes of immunoglobulins places a premium on efforts to adapt the IgG-tailored experimental strategies to other classes of antibodies, but their dramatically higher levels of glycosylation may create insurmountable obstacles. The monoclonal murine IgE antibody explored in this work provides a challenging model system, as its glycosylation level exceeds that of conventional IgG mAbs by a factor of nine. The commercial sample, which included various IgE fragments, yields a poorly resolved ionic signal in intact-mass measurements, from which little useful information can be extracted. However, coupling MS measurements with the limited charge reduction of select polycationic species in the gas phase gives rise to well-defined charge ladders, from which both ionic masses and charges can be readily determined. The measurements reveal significant variation of the extent of glycosylation within intact IgE molecules, as well as the presence of low-molecular weight impurities in the commercial IgE sample. Furthermore, incubation of the monoclonal IgE with its antigen (ovalbumin) gives rise to the formation of complexes with varying stoichiometries, which can also be uniquely identified using a combination of native MS, limited charge reduction in the gas phase and data fitting procedures. This work demonstrates that following appropriate modifications, intact-mass analysis measurements can be successfully applied to mAbs beyond the IgG isotype, providing a wealth of information not only on the mass distribution of the intact IgE molecules, but also their large-scale conformational integrity, the integrity of their covalent structure, and their interactions with antigens.
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影响因子：
4.4
作者：
Barnidge, David R.;Dasari, Surendra;Murray, David L.
通讯作者：
Murray, David L.
DOI：
10.1021/jasms.0c00461
发表时间：
2021-06-02
影响因子：
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作者：
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作者：
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DOI：
10.1007/s13361-017-1781-1
发表时间：
2017-12
影响因子：
3.2
作者：
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影响因子：
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作者：
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